Transformed Shigella

ABSTRACT

A method for modifying a wild strain of an entero-invasive  Shigella  to produce a modified strain of  Shigella  that can be used for making a vaccine against the wild strain of  Shigella . The genome of the wild strain of  Shigella  is transformed so that it cannot substantially invade cells of a human host and cannot spread substantially within infected cells and from infected to uninfected cells of the host and cannot produce toxins which will kill substantial numbers of the host&#39;s infected, as well as uninfected, cells. A first gene of the wild strain of  Shigella , coding for a protein necessary for the  Shigella  to invade cells of the host, and a second gene, coding for a protein necessary for the  Shigella  to spread within infected cells and between the infected and uninfected cells of the host, are mutagenized.

This is a continuation of application Ser. No. 08/466,698, filed Jun. 6,1995 (abandoned), itself a continuation of application Ser. No.08/118,100, filed Sep. 8, 1993 (Now U.S. Pat. No. 5,762,941), which is acontinuation of application Ser. No. 07/460,946, filed Mar. 21, 1990(abandoned), which is the national phase of PCT/EP89/00831, filed Jul.14, 1989, published as WO90/00604, Jan. 25, 1990, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to a method of modifying the genome of anentero-invasive wild strain of Shigella so that the strain cannotsubstantially invade cells and tissues of an infected host and cannotspread substantially within infected cells and between infected andnon-infected cells of the host and cannot produce toxins which will killsubstantial numbers of the hosts' cells. This invention particularlyrelates to such a modified strain of Shigella which can be used toimmunize a host against the wild strain of Shigella.

Shigellosis or bacillary dysentery is a disease that is endemicthroughout the world. The disease presents a particularly serious publichealth problem in tropical regions and developing countries whereShigella dysenteriae 1 and S. flexneri predominate. In industrializedcountries, the principal etiologic agent is S. sonnei although sporadiccases of shigellosis are encountered due to S. flexneri, S. boydii andcertain entero-invasive Escherichia coli.

The primary step in the pathogenesis of bacillary dysentery is invasionof the human colonic mucosa by Shigella (23). Mucosal invasionencompasses several steps which include penetration of the bacteria intoepithelial cells, intracellular multiplication, killing of host cells,and final spreading to adjacent cells and to connective tissue (9, 41,55, 56). The overall process which is usually limited to the mucosalsurface leads to a strong inflammatory reaction which is responsible forabscesses and ulcerations (23, 41, 55).

Even though dysentery is characteristic of shigellosis, it may bepreceded by watery diarrhea. Diarrhea appears to be the result ofdisturbances in colonic reabsorption and increased jejunal secretionwhereas dysentery is a purely colonic process (20, 41). Systemicmanifestations may also be observed in the course of shigellosis, mainlyin the cases due to S. dysenteriae 1. These include toxic megacolon,leukemoid reactions and hemolytic-uremic syndrome (“HUS”). The latter isa major cause of mortality from shigellosis in developing areas (11, 22,38).

The role of Shiga-toxin produced at high level by S. dysenteriae 1 (6)and Shiga-like toxins (“SLT”) produced at low level by S. flexneri andS. sonnei (19, 30) in the four major stages of shigellosis (i.e.,invasion of individual epithelial cells, tissue invasion, diarrhea andsystemic symptoms) is not well understood. For review see O'Brien andHolmes (32). Plasmids of 180–220 kilobases (“kb”) are essential in allShigella species for invasion of individual epithelial cells (41, 42,44). This includes entry, intracellular multiplication and early killingof host cells (4, 5, 46). The role of Shiga-toxin and SLT at this stageis unclear. They do not appear to play a crucial role in intracellularmultiplication and early killing (4, 12, 46). However none of theexperiments which have been carried out has compared isogenic mutants ina relevant cell assay system. Recent evidence indicates that Shiga-toxinis cytotoxic for primary cultures of human colonic cells (27). Tissueinvasion requires additional chromosomally encoded products among whichare smooth lipopolysaccharides (“LPS”) (44, 57), the non-characterizedproduct of the Kcp locus (8, 44), and aerobactin (24, 28). A region ofthe S. flexneri chromosome necessary for fluid production in rabbitileal loops has been localized to the rha-mtl regions and near thelysine decarboxylase locus (44). However, no evidence has been adducedto show that the ability to cause fluid accumulation is due to the SLTof S. flexneri. Thus, the role of Shiga-toxin in causing the systemiccomplications of shigellosis is still hypothetical. However, Shiga-toxincan mediate vascular damage since capillary lesions observed in HUSresemble those observed in cerebral vessels of animals injected withthis toxin (1, 2, 22).

A mutant which lacks Shiga-toxin or SLT could indicate the role of thesetoxins in the disease process. S. dysenteriae 1, which produces thehighest amount of this cytotoxin, could be transformed into such aShiga-toxin negative mutant (“Tox−”) and could serve best to indicatethe role of the toxin—despite Sekizaki et al's (48) having obtained sucha mutant which appeared as invasive in the HeLa cell assay and theSereny test (49) as the wild strain. More importantly, such a Tox⁻mutant could be used to make a mutant which could not invade, and thenmultiply substantially within, cells of a host and also could not spreadsubstantially within the host's infected cells and from there to thehost's uninfected cells and also could not produce toxins which wouldkill subtantial numbers of infected, as well as uninfected, host cells.As a result, the Tox⁻ mutant could be used to immunize a host against awild strain of the Shigella.

SUMMARY OF THE INVENTION

A Tox⁻ mutant of a wild strain of S. dysenteriae 1 is geneticallyengineered by allelic exchange with an in vitro mutagenized Shiga-toxingene. The effect of this mutation in cell assay systems and animalsshows that the mutant can be genetically engineered further to provide amutant which cannot substantially invade and then spread within andbetween host cells and cannot produce Shiga-toxins in host cells.

Also in accordance with the invention, the Tox⁻ mutant of the wildstrain of S. dysenteriae 1 is genetically engineered further by allelicexchange with:

-   -   a) an in vitro mutagenized gene of S. dysenteriae 1 which        encodes a protein necessary for S. dysenteriae 1 to invade a        host's cells, as well as tissues, such as a gene which codes for        a protein necessary for the chelation of iron and/or the        transport of iron into S. dysenteriae 1 (e.g., an enterobactin        or enterochelin gene of S. dysenteriae 1); and    -   b) an in vitro mutagenized gene of S. dysenteriae 1 which        encodes a protein necessary for S. dysenteriae 1 to spread        within infected cells and between infected and uninfected cells,        such as an intra-intercellular spread gene (e.g., an ics A or        vir G gene).

Further in accordance with this invention, a mutant of a wild strain ofS. flexneri is genetically engineered by allelic exchange with: a) an invitro mutagenized gene of S. flexneri which encodes a protein necessaryfor S. flexneri to invade a host's cells, as well as tissues, such as agene which codes for a protein necessary for the chelation of ironand/or the transport of iron into S. flexneri (e.g., an aerobactin geneof S. flexneri); and b) an in vitro mutagenized gene which encodes aprotein necessary for S. flexneri to spread within and between thehost's cells, such as an ics A gene.

Still further in accordance with this invention, the mutants of Shigellaof this invention are used for making vaccines against the wild strainsof Shigella.

BRIEF DESCRIPTION OF THE FIGURE

The Figure shows schematically the cloning of the Shiga-toxin operon andin vitro mutagenesis of the Shiga-toxin A subunit gene in Example 2. Inplasmids pHS7201, pHS7202 and pHS7203 in the Figure: Solid linesindicate sequences from the A subunit gene; Stippled lines indicate Bsubunit gene sequences; and Stripped lines indicate sequences from the Ωinsertion element.

DETAILED DESCRIPTION OF THE INVENTION

A method is provided for modifying a wild strain of an entero-invasiveShigella so that the modified strain can be used for making a vaccineagainst the wild strain of Shigella. The wild strain of Shigella ismodified so that it cannot invade and then multiply substantially withininfected cells of a host, particularly a human host, and cannot spreadsubstantially within infected cells and from infected to uninfectedcells of the host and cannot produce toxins which will kill substantialnumbers of the host's infected, as well as uninfected, cells. The methodinvolves transforming the genome, (e.g., the large virulence plasmidpHS7200) of the wild strain of Shigella, such as an S. flexneri, so thatgene(s) of the wild strain, coding for one or more proteins necessaryfor the strain to invade an infected host's cells, as well as tissues(e.g., an aerobactin gene), and coding for one or more proteinsnecessary for the strain to spread within and between the infectedhost's cells (e.g., an ics A gene [60, 61]), are wholly or partlyremoved or permanently inactivated, preferably at least partly removed.For transforming the genome of a wild strain such as a S. dysenteriae 1,the method preferably involves also wholly or partly removing orpermanently inactivating, preferably at least partly removing, thegene(s), preferably just the A subunit gene, coding for Shiga-toxin.

In the method of this invention, the genes of the wild strain ofShigella can be wholly or partly removed or permanently inactivated in aconventional manner, for example by allelic exchange with in vitromutagenized genes, at least significant portions of which preferablyhave been removed. In this regard, it is preferred that the mutagenizedgenes not be simply inactivated by means of transposons which areinserted into the genes and which can be lost by the genes when they arereproduced in vivo in subsequent Shigella generations when makingvaccines of this invention. Rather, the mutagenized genes preferablyhave had significant portions thereof deleted, and suitablevaccine-compatible marker genes are preferably inserted within suchdeletions. Such marker genes permit so-transformed Shigella to be easilyidentified. The preferred marker genes are the heavy metal-resistancegenes such as the mercury, arsenate, arsenite, antimony, cadmium, zincand/or cobalt-resistance genes (62, 63, 64, 65).

The cells of the modified strain can be cultured and then attenuated ina conventional manner. The cells can then be mixed with conventionalpharmaceutically acceptable vehicles (e.g., an aqueous saline solution)and optionally with conventional excipients (e.g., a pharmaceuticallyacceptable detergent) to form a vaccine against the wild strain. Thevaccine can be formulated to contain a final concentration of cellmaterial in the range of 0.2 to 5 mg/ml, preferably 0.5 to 2 mg/ml.After formulation, the vaccine can be incorporated into a sterilecontainer which is then sealed and stored at a low temperature (e.g., 4°C.), or it can be freeze dried.

In order to induce immunity in a human host to a wild strain ofShigella, one or more doses of the vaccine, suitably formulated, can beadministered in doses containing about 10⁹–10¹¹ lyophilized Shigellacells. The vaccine can be administered orally in a conventional manner.The treatment can consist of a single dose of vaccine or a plurality ofdoses over a period of time.

The Examples, which follow, illustrate this invention.

EXAMPLES

Unless otherwise indicated, the cloning and transformation proceduresand techniques used in the Examples are the same as are generallydescribed in Maniatis et al, “Molecular Cloning—A Laboratory Manual”,Cold Spring Harbor Laboratory (1982).

The strains, used in Example 1–6, and their phage or plasmid content areset forth in Table I.

Two media were used in the Examples: M9 minimal medium (Na₂HPO₄.12H₂O:15 g/l, KH₂PO₄: 3 g/l, NaCl: 0.5 g/l, NH₄Cl: 1 g/l, MgSO₄.7H₂O: 0.05g/l) and Trypto Casein Soja Broth (Diagnostics Pasteur, Marnes 1aCoquette, France).

Example 1 Cloning of the Shiga-Toxin Operon

Total DNA was prepared (50) from a wild type antibiotic-sensitive S.dysenteriae 1 strain SC500 obtained from Centre National de Référencedes Shigelles of Institut Pasteur, Paris, France. 10 μg of DNA weredigested with EcoRI (Amersham, Buckinghamshire, UK) and loaded on a 0.7%agarose gel. Fragments ranging from 3.5 to 4.5 kb were electroeluted.0.1 μg of purified fragments was ligated to 1 μg of cos-ligated, EcoRIcut, dephosphorylated λ GT11 arms (Stratagene Cloning System, San Diego,USA) and packaged using Packagene System (Progema Biotec, Madison, USA)according to the suppliers recommendations. The packaged DNA was thentransfected into E. coli Y1090(59). The λ GT11 bank was then screenedwith 13C4, a monoclonal antibody specific for the B subunit of SLT1 (54)obtained from A. D. O'Brien, U.S.U.S.H., Bethesda, Md., USA. 10³recombinant phages were plated on Y1090 in LB soft agar. Plates wereincubated at 37° C. for 12 hours. A nitrocellulose filter (Schleicherand Schüll, Dassel, FRG), previously dipped into a 10 mMisopropylthiogalactoside (“IPTG”) solution (Sigma, St Louis, Mo., USA)was applied to the plate which was then incubated at 42° C. for 2.5hours. The filter was removed from the plate and incubated 1 hour at 37°C. in PBS-milk (50 g/l dehydrated low-fat milk in 1×PBS), washed fivetimes with 1×PBS, and incubated for 1 hour with the 13C4 monoclonalantibody in its non-diluted hybridoma cell supernatant. After fivewashes in PBS-milk, the filter was incubated 1 hour at 37° C. inPBS-milk containing a 1/200 dilution of sheep anti-mouse IgG antibodyconjugated with alkaline phosphatase (Biosys, Compiègne, France). Thefilter was washed again in 1×PBS and placed in the staining solution:0.33 mg/l nitro-blue tetrazolium, 0.16 mg/l 5-bromo-4-chloro-3-indolylphosphate (both compounds from Sigma), 100 mM Tris HCl pH 9.5, 100 mMNaCl, 50 mM MgCl₂. Positive clones were plaque purified and transfectedinto Y1089 (59). DNA was then prepared from the lysogen (13). Subcloningwas done in the EcoRI site of plasmid vector pUC8 in E. coli JM83 (58).Subclones of E. coli JM83 were tested with monoclonal antibody 13C4 asdescribed above with the following modifications: a dry nitrocellulosefilter was applied onto the plate and 2 ml of a 2 mg/l polymyxin Bsolution in PBS were added on top of the filter. The plate was thenincubated at 37° C. for 45 minutes before starting PBS-milk incubation.Subclone pHS7201 in E. coli JM83, containing the B subunit of SLT1, wasidentified.

Subclone pHS7201 of E. coli JM83 was found to have a stronger signal incolony immunoblot assay in the presence of 13C4 monoclonal antibody thanparental strain SC500 due to the gene dosage effect. A restriction mapof the Shiga-toxin coding region within pHS7201 was identical to that ofSLT1 (14). The A subunit gene was seen to possess a unique Hpal sitelocated 310 bp downstream from the ATG starting codon where a cassettecould be inserted as described in Example 2.

Example 2 In vitro Mutagenesis of the Shiga-Toxin A Subunit Gene

In subclone pHS7201, the entire Shiga-toxin operon is contained in a 4.2kb EcoRI DNA fragment. In vitro mutagenesis of the A subunit gene wasdone,by inserting the interposon Ω(37) which codes for spectinomycinresistance and is flanked on each side by T4 translation transcriptionstop-signals. Ω was purified as an HindIII 2 kb fragment, and its endswere filled in by the Klenow fragment of DNA polymerase I. Ω was thenligated to HpaI linearized pHS7201 to generate the recombinant plasmidpHS7202 as shown in the Figure. The 6.2 kb EcoRI fragment containing themutagenized sequence was then purified and ligated with the EcoRI siteof the suicide plasmid vector pJM703.1 (51) to generate recombinantplasmid pHS7203 as shown in the Figure. pJM703.1 replicates only if itsdeficient R6K origin is complemented in-trans by the pir functioncontained in the lambda phage integrated in the genome of E. coli SM10(21). This strain also contains the transfer genes of the broad hostrange IncP-type plasmid RP4 integrated in its chromosome. pJM703.1 canthus be mobilized by SM10 λ pir (21) because it contains the Mob sitefrom RP4 (51). pHS7203 was thus stably maintained in strain SM10 λ pirand was then conjugally transferred into wild type S. dysenteriae 1strain SC500. Matings were performed on cellophane membranes, selectionwas obtained by plating on M9 minimal medium supplemented with thiamine,methionine, tryptophan and nicotinic acid at a concentration of 10 μg/mleach, 0.2% glucose and 50 μg/ml spectinomycin. Colonies growing onselective medium were purified and identified as S. dysenteriae 1 byagglutination with a specific rabbit antiserum (Diagnostics Pasteur).

Allelic exchange between the wild-type chromosomal Shiga-toxin gene andthe in vitro mutagenized gene of Shiga-toxin was shown by colony blotimmunoassay, using the monoclonal antibody 13C4 to detect S. dysenteriae1 cells expressing a Tox-phenotype.

The presence of the Tox⁻ modification in the genomes of the S.dysenteriae 1 cells was verified with a probe made from the 655 bpHindIII-HincII fragment containing part of the A subunit gene and theentire B subunit gene from the 4.2 kb EcoRI fragment, described above,containing the entire Shiga-toxin operon. The 2 kb HindIII fragment,described above, containing the Ω interposon, was also used as a probe(37). The DNA fragments, used as the probes, were labeled bynick-translation (39) with ³²p-labeled 5′-dCTP (Amersham). Total DNA wasprepared from two Tox-clones and analyzed by hybridization with theShiga-toxin probe and the Ω probe. The DNA fragments were transferredfrom agarose gels to nitrocellulose filters (Schleicher and Schüll) bythe method of Southern (53). Hybridization was carried out at 65° C.overnight, and washing was done at 65° C. in 6×SSC. The probes showedthat the 4.2 kb EcoRI fragment from S. dysenteriae 1 containing thetoxin genes had been replaced in the Tox-mutants by the 6.2 kb fragment,which hybridized with both probes. This result showed that the flankingregions on each side of the mutagenized toxin gene in pHS7203 hadrecombined with their counterparts in the SC500 genome, thus replacingthe wild-type A subunit gene by the mutated gene.

One of these Tox-clones, SC501, was selected for further study, andclone SC501 was deposited with the Centre Nationale de Cultures deMicroorganismes of Institut Pasteur, Paris, France, under accession no.I-774, on Jun. 30, 1988.

Example 3 Assays of Cytotoxicity, Growth within HeLa Cells, MacrophageDetachment and Toxicity in Rabbit Ileal Loop and in Monkey

SC500 and SC501, as well as their non-invasive derivatives SC502 andSC503 respectively (obtained by the spontaneous-cure (i.e., loss) oftheir large virulence plasmid pHS7200 which is necessary for invasion ofcells), were grown for 48 hours in 200 ml of iron-depleted medium.Glassware was pretreated with 6N HCl and rinsed extensively with ironfree H₂O . The medium contained M9 salts supplemented with 15 μg/mlCaCl₂, 5 mg/ml casamino-acids, 2 mg/ml glucose, 50 μg/ml thiamine, 20μg/ml L-tryptophane, 10 μg/ml nicotinic acid and 150 μg/ml humantransferrin (Sigma). The bacteria were washed twice in saline andresuspended in 3 ml of PBS. Lysozyme was added at a final concentrationof 0.2 mg/ml. After a 30 minute-incubation at room temperature (25° C.),30 μl EDTA 0.5 M pH8 was added, and the cells were transferred to an icebath and sonicated. Sonic extracts were filter-sterilized and keptfrozen at −20° C. Filter sterilized culture supernatants and bacterialextracts were assayed for cytotoxicity on HeLa cells grown in minimalessential medium with Earle's salts and N-glutamine (Gibco, Paisley,Scotland, UK) supplemented with 10% foetal calf serum (Gibco). Serialdilutions were made in cell culture medium (100 μl) in a microtitierplate. Each well was inoculated with 2×10⁴ cells in 100 μl. Plates werethen incubated at 37° C. in 5% CO₂ for 24 hours. Neutralization assayswere performed both with a rabbit polyclonal serum and the 13C4monoclonal antibody. Plates were examined under light phase microscopy,then stained with Giemsa. Cytotoxicity was calculated as the cytotoxicdose 50% (CD50) per mg of protein of the extract.

Multiplication of bacteria within HeLa cells was assayed (46).Non-confluent monolayers of HeLa cells in 35 mm plastic tissue culturedishes (Becton Dickinson Labware, Oxnard, Calif., USA) were inoculatedwith bacteria, resuspended in 2 ml of minimum essential medium (“MEM”,Gibco) at a multiple of infection (“MDI”) of 100, centrifuged for 10minutes at 2,200×g and incubated for 30 minutes at 37° C. to allowentry. Plates were then washed three times with Earle's Balanced SaltSolution (“EBBS”, Gibco) and covered with 2 ml of MEM with gentamicin(25 μg/ml). This was defined as time 0 (To). After one hour ofincubation at 37° C., preparations were washed again, with EBSS andcovered with 2 ml of MEM without antibiotic (T1). Incubation wascontinued for three more hours (T1–T4). Two plates were removed everyhour. One plate was washed three times with EBSS and Giemsa stained tocalculate the percentage of infected HeLa cells. The other was washedfive times with EBSS to eliminate viable extracellular bacteria. Cellswere trypsinized, counted and lysed at 0.5% sodium deoxycholate indistilled water. Dilutions were plated onto Trypticase Soy Agar. Theaverage number of bacteria per infected HeLa cell was calculated.Experiments were repeated four times. Intracellular growth curves weredrawn and the slope at exponentional phase was calculated.

Assay for macrophage detachment and killing was performed (4) using J774macrophages (52) maintained in RPMI 1640 (Flow Laboratories Inc.,McLean, Va., USA) supplemented with complement-inactivated foetal calfserum (Gibco) and 2 mM glutamine (Gibco). Eighteen hours beforeinfection, 7×10⁵ macrophages in 35 mm plastic tissue culture dishes(Becton Dickinson Labware) were labeled in a culture medium. containing0.5 μCi of [³H] uridine per ml (Amersham). Cells were washed three timeswith EBSS before addition of 1 ml of the bacterial suspension in RPMI1640 at a MOI of 100. Infection was performed for one hour at 37° C. in5% CO₂. Monolayers were then washed three times with EBSS (To) andcovered for one hour at 37° C. in 5% CO₂ with 2 ml of RPMI supplementedwith 2 mM glutamine and gentamicin 25 μg/ml (T1). Plates were thenwashed three times with EBSS and incubated in 5% CO₂ for 3 more hours(T1–T4) at 37° C. in RPMI glucose without gentamicin. Two plates wereremoved every hour, cultures were washed three times with EBSS and thepercentage of non viable macrophages among cells that still adhered tothe plastic surface was determined by trypan blue staining. Thepercentage of residual macrophages was then determined by measuring theamount of radioactivity remaining in the dish. Adherent cells were lysedwith 1 ml of 0.5% sodium deoxycholate in distilled water and 100 μl ofthis lysate was precipitated and counted (4).

Rabbit ligated ileal loops of 10 cm were prepared in rabbits of ca. 2 kgwhich were anesthesized with 0.5 ml/kg of 6% sodium pentobarbital.Inocula of 10⁷ and 10⁹ CFU in 1 ml of Trypticase Soy Broth were tested.Rabbits were sacrificed 18 hours later. Fluid accumulation within loopswas recorded, and the volume-to-length ration (“V/L”) was calculated.Portions of infected loops were fixed in 10% buffered formalin.Specimens were processed by standard procedures and stained withhematoxylin-eosin-safranin.

Eight rhesus monkeys weighing 3.5 to 4.5 kg were injectedintramascularly with 50 mg of ketamine chlorhydrate (Imalgene 500, RhôneMérieux, Lyon, France). Each animal was inoculated intragastrically with1.5×10¹¹ of SC500 and SC501 microorganisms resuspended in 20 ml ofTrypticase Soy Broth and 14 g/l sodium bicarbonate (50/50). Plating ofthe inoculum on Congo-red agar indicated that less than 1% of thebacteria in the inoculum had lost their invasive property (26). Stoolswere examined daily for diarrhea, presence of pus, mucus and blood.Intensity of each of these symptoms was graded from 0 to 3+ every day.For each animal, the severity of a given symptom was expressed as anindex which represented a sum of the accumulated “+” for each symptom.Immediate autopsy was performed in monkeys who died of fulminantdysentery. Species ware processed as described above for rabbit tissues.

Results

SM10 λ pir (pHS7203) was noncytotoxic in the cytotoxicity assay. Afterconjugative transfer of pHS7203 into S. dysenteriae, clones thatdisplayed the Amp^(S) Spc^(R) phenotype were tested in the colonyimmunoblot assay. Five per cent displayed a Tox⁻ phenotype. SC501 showeda cytotoxicity of 347 CD50/mg of protein, which was the same order ofmagnitude as that of well-known E. coli K12 (412 CD50/mg). Residualcytotoxicity from SC501 could not be neutralized by an anti-Shiga-toxinpolyclonal serum.

The presence of the Tox-mutation in strain SC501 did not significantlyalter its capacity to grow intracellularly within HeLa cells since itsrate of exponential growth, expressed in generations/hour, was 2.6±0.7compared to 2.5±0.6 for wild-type strain SC500. In addition, nosignificant difference could be observed in the efficiency of rapidkilling of J774 macrophages by SC500 and SC501. Both cell detachment andappearance of Trypan Blue positive cells progressed at similar ratesover four hours, thus indicating that Shiga-toxin released withininfected cells neither significantly affected the rate of intracellulargrowth nor increased rapid killing of host cells.

The effect of the Inv⁻ and Tox⁻ mutations on the pathogenicity of S.dysenteriae 1 in the rabbit ligated loop model was determined by theeffect on fluid production within loops. Mean and standard deviationswere computed from the results obtained in six loops for each strain ateither of the two inocula (i.e., 10⁹ and 10⁷ CFU). For invasive strains(i.e., SC500, Inv⁺, Tox⁺ and SC501, Inv⁺, Tox⁻) at both inocula, thelack of Shiga-toxin production decreased fluid accumulation, but thedifference was not statistically significant, indicating that invasionand subsequent inflammation are primarily responsible for fluidaccumulation. For non-invasive strains (i.e., SC503, Tox⁺ and SC502,Tox⁻) a striking difference was observed since only the strain producingShiga-toxin elicited fluid accumulation. This indicated that, in therabbit model, Shiga-toxin is the only enterotoxin of S. dysenteriae 1,whatever the role this enterotoxin may play in the course of thedisease. Histopathological studies showed severe lesions includingabscesses and ulcerations destroying numerous villi at both inoculaeither with SC500 or SC501. In general, lesions were more severe inloops infected with the wild-type strain, but the observation that thedifference was minor indicated that invasion was the major factor ofpathogenecity.

Loops infected with SC502, the non-invasive Tox⁺ strain, were severelyalterated with swelling and shortening of the villi, oedema andinflammation of the lamina propria, alterations of epithelial cells withlarge amounts of mucus shed from goblet cells and areas of killedenterocytes with pycnotic nuclei. However, the most striking feature washemorrhages throughout the epithelial layer.

The effect of the Tox⁻ mutation on the pathogenicity of S. dysenteriae 1was shown in monkeys. Two animals died of fulminant dysentery at day 4in both the group injected with SC500 and the group injected with SC501,each group thus indicating that Shiga-toxin was not required for lethaldysentery. No significant differences could be observed in the volume ofdiarrheic stools and the amount of pus and mucus, although the latterwere difficult to quantify with precision. On the other hand, thepresence of blood was a constant characteristic of abnormal stools inanimals infected with SC500 whereas only one animal infected with SC501showed transcient presence of a slight amount of blood. Autopsiesperformed immediately after the death of the animals showed obviousdifferences in the colonic peritoneal mesothelium which was particularlyapparent on the surface of the sigmoid on which patchy hemorrhagic areascould be observed only in the case of animals infected with SC500. Onthe average, the number and severity of abscesses was similar, butpurulent necrosis of the mucosa with destruction in Lieberkühn glandswas only observed, in some areas, in animals infected with SC500.Inflammatory infiltration of the chorion, submucosal tissues andperitoneum was also more severe in these animals. In addition, theinflammatory infiltrate of the peritoneal mesothelium which wascharacteristic of animals infected with SC500 as compared to SC501, waspredominantly perivascular thus confirming the gross examination whichsuggested the presence of a severe peritoneal vasculitis. However, themost striking difference was observed at the level of the capillarycirculation within the interglandular chorion. Monkeys infected withSC500 showed hemorrhages disrupting the structure of the upper part ofthe mucosa. Erythrocytes could be observed being released into theintestinal luman through microabscesses which caused local interruptionof the epithelial lining. These hemorrhages were obviously due todestruction of the capillary loops. On the other hand, monkeys infectedwith SC501 showed dilatation of the capillary loop but no disruption.White blood cell counts performed before and at day 3 after infectionshowed: at day 0, no significant difference in polymorpho nuclear cell(“PMN”) counts, and myelemia was absent; and at day 3, the drop in bloodPMN and the level of myelemia were each more pronounced in monkeysinfected by SC500.

Conclusions

Circumstantial evidence in humans supports the hypothesis thatShiga-toxin is a true virulence factor. Volunteers fed strain 725, aninvasive, low-toxin producing, chlorate-resistant mutant of S.dysenteriae 1, showed less severe symptoms than those fed the wild-typestrain M131 (25). Patients experiencing natural infection usuallydevelop more severe symptoms including HUS when infected with S.dysenteriae 1 than with other Shigella serotypes (7). They rapidlydevelop toxin-neutralizing antibodies (18).

The Tox⁻ mutant of S. dysenteriae 1, SC501, has been shown to produce aresidual amount of cytotoxin similar to E. coli K12. This mutant hasbeen used to study the role of this Shiga-toxin in the virulence of S.dysenteriae 1. Cellular assays and more definitive animal models havebeen used.

Assays using HeLa cells and J774 macrophages in monolayers have shownthat secretion of Shiga-toxin did not affect the rate of exponentialgrowth within infected cells as suggested for SLT in S. flexneri in aprevious study (46). These results were in agreement with theobservation that two other low toxin producer mutants (25, 48) as wellas the SC501 mutant do not affect keratoconjuctivitis (49) which isknown to correlate with the capacity of bacteria to multiply within anepithelium (35). As also suggested previously (4, 12), no correlationcould be observed between Shiga-toxin production and early killing ofhost cells. Although such data need confirmation in assays that wouldmore closely mimic the actual infection, they certainly indicate thatShiga-toxin does not play a major role at the intracellular stage ofinfection. Invasion appears to trigger early metabolic events whichmediate killing of host cells (47) more rapidly than the slow actingprocess of Shiga-toxin (12).

Infection of rabbit ligated intestinal loops demonstrated only slightdifferences in the severity of mucosal lesions after 18 hours with boththe SC500 and SC 501 inocula. However, the duration of exposure andclosing of loops may mask the effect of cytotoxin production and makeinvasion the primary event. Results concerning enterotoxicity were moredifficult to analyze in the case of invasive bacteria since the amountof fluid produced, although lower at both inocula for the Tox⁻ mutant,was not significantly different from that. elicited by the wild-typestrain. This indicated that invasion of tissues is sufficient to blockthe reabsorbative functions of the epithelium. On the other hand, thestriking difference observed between non-invasive Tox⁺ and Tox⁻ mutantsindicates that, within the limits of sensitivity of the rabbit model,Shiga-toxin is the only enterotoxin of S. dysenteriae 1. This is inagreement with previous studies (16, 17, 33). However, when observingfluid production by Inv⁺ and Inv⁻ mutants, the nature of the fluidproduced varies according to the infecting strain. Invasive strainselicit production of a viscous, mucopurulent, sometimes bloody liquidwhich probably reflects the extent of abscesses ulcerated within thelumen regardless of the amount of Shiga-toxin produced, whereasnon-invasive, Tox⁺ strains produce a watery, sometimes bloody, liquidwhich is more a reflection of enterotoxicity and cytotoxicity.Histopathological studies of tissue samples from loops infected withSC502, the Inv⁻, Tox⁺ mutant, showed an important inflammatoryinfiltrate of the lamina propria and major alterations predominantly atthe tip of shortened villi. This confirmed the cytotoxicity ofShiga-toxin on enterocytes in vivo (27). However, the most strikingfeature was infiltration of the epithelial lining by erythrocytes whichwere shed into the lumen along with important amounts of mucus. Thisobservation, which suggested that major vascular alterations hadoccurred within the lamina propria, was subsequently confirmed in themonkey model.

Intragastric inoculation of SC500 and SC501 in macaque monkeysdemonstrated that lethal fulminant dysentery could occur regardless ofShiga-toxin production. No significant difference was observed in theamount of diarrhea, pus and mucus in stolls. Absence of watery diarrheaand equal amount of stool were not consistent with previous studiessuggesting increased jejunal secretion bu Shiga-toxin (41). The onlystriking difference was the presence of blood in dysenteric stools ofanimals infected with the wild type strain. A recent paper reportedthat, among patients presenting shigellosis, those who eliminatedstrains of higher cytotoxicity were more likely to present blood intheir stools (36). Histopathological observations confirmed the presenceof vascular damages which appeared particularly characteristic in thesigmoid since monkeys infected with the wild type strain showed totaldestruction of the capillary loops within the chorion whereas thevascular system of animals infected with the Tox⁻ mutant showedturgescent but mostly intact vessels. This certainly explains thepresence of bloody stools in the former group. In addition, observationof the peritoneal mesothelium showed oedema and severe inflammatoryvasculitis. Thus, release of Shiga-toxin by invading bacteria within thetissues may locally enhance severity of the mucosal lesion by evokinglocal ischemia through destruction of the chorion blood flow andalterations of the peritoneal as well as possibly mesentericcirculation. This effect appears to be local or loco-regional sinceobservation of kidney tissues did not show evidence of capillaryvasculitis at this stage of the disease (data not shown). Such vascularalterations may be consistent with observations in hemorrhagic colitisdue to E. Coli 0157:H7 (40) in which a radiologic aspect of ischemiccolitis has been described (34). These strains produce high levels ofSLT1 (31) which has a direct cytopathic effect on dividing endothelialcells (15).

Another difference observed between animals infected with Tox⁺ and Tox⁻strains was the severity of mucosal inflammation and subsequentabscesses. In many areas of the sigmoid and transverse colons, lesionsappeared of similar intensity, but only animals infected with SC500showed areas with impressive purulent destruction of mucosal tissues.

Higher intensity of the purulent exsudate was reflected in a moredramatic drop of blood PMN with consecutive myelemia at day three ofinfection. It is believed that, in addition to the marrow and vascularcompartments, a third PMN compartment is opened at the colonic levelduring shigellosis. Shiga-toxin is expected to increase the number ofPMN entrapped within this new compartment through vascular alterationswhich increase diapedesis as well as direct release of PMN withinmucosal tissues. This would account for the rapid and severegranulocytopenia observed in animals infected by the wild type strainand for subsequent higher myelemia which may be an equivalent of theleukemoid reaction sometimes observed in the course of severeshigellosis. Such a model does not postulate a systemic effect ofShiga-toxin.

The foregoing results thus suggest that Shiga-toxin plays a limited rolewhen released intracellularly within epithelial and phagocytic cells.However, Shiga-toxin released within infected tissues appears to actpredominantly through intestinal vascular damage.

Example 4

Using the procedure of Example 2, SC501 is genetically engineered by invitro mutagenesis of its operon coding for enterochelin. The suicideplasmid vector pJM703.1, that is utilized, contains the enterochelinoperon of S. dysenteriae 1, with each of its ent F, Fep E, Fep C and FepD subunit genes mutagenized with an interposon which codes forresistance to the herbicide Biolafos and a suitable promoter for theherbicide resistance gene. The resulting clone, SC504, is Tox⁻ andenterochelin⁻ (“Ent⁻”).

Example 5

Using the procedure of Example 2, SC504 is genetically engineered by invitro mutagenesis of its ics A gene. The suicide plasmid vectorpJM703.1, that is used, contains the ics A gene of S. flexneri (60, 61),which has been mutagenized with an interposon. The resulting clone,SC505, is Tox⁻, Ent⁻ and ics A⁻ and can be used in making a vaccineagainst S. dysenteriae 1.

Example 6

Using the procedure of Example 2, a wild type S. flexneri is geneticallyengineered by in vitro mutagenesis of its gene coding for aerobactin andits ics A gene. The suicide plasmid vector, that is used, contains theaerobactin and ics A genes of S. flexneri which have each beenmutagenized with an interposon. The resulting clone, SC506, isaerobactin⁻ and ics A⁻ and can be used in making a vaccine against S.flexneri.

Example 7

Using the procedure of Examples 1, 2 and 4, a 400 basepair Bal31deletion is made, starting from the unique Hpa1 site, inside the Asubunit gene of the Shiga-toxin operon in a DNA fragment from S.dysenteriae 1 in strain SC500. The resulting fragment is religated witha 257 basepair fragment containing the P1 promoter of pBR322, thusallowing high expression of the B subunit protein. This fragment,containing the mutagenized toxin A gene, is cloned into a conditionalsuicide vector which contains a replication of origin under the controlof the E. coli lac promoter and a kanamycin resistance gene. In S.dysenteriae 1, this vector will replicate only if IPTG is present in theculture medium. A mercury-resistance cartridge (65) is inserted upstreamfrom the mutagenized A subunit gene. The resulting plasmid istransformed into the wild type S. dysenteriae 1 strain SC500 in thepresence of IPTG. Colonies of the resulting Shigellaclone are Hg andkanamycin resistant. They are allowed to grow for many generations inthe absence of IPTG. The cultures are then screened. for the presence ofHg-resistant kanamycin-sensitive clones. Three clones are isolated andfurther characterized. Southern blots show that they no longer hybridizewith an A subunit gene internal probe but still produce high amounts ofB subunit protein, as detected by monoclonal antibody analysis, and theyno longer are cytotoxic.

Using the same procedure, this ToxA⁻ clone is genetically engineered byin vitro mutagenesis of its operon coding for enterochelin. The suicideplasmid vector, that is utilized, contains the enterochelin operon of E.coli (66), with each of its ent F, Fep E, Fep C and Fep D subunit geneshaving a significant deletion at a restriction site, into which isinserted a fragment that codes for resistance to arsenite (62) and asuitable promoter for the arsenite-resistance gene. The resulting cloneis Tox A⁻ and Ent⁻.

Using the same procedure, this Tox A⁻ and Ent⁻ clone is geneticallyengineered by in vitro mutagenesis of its ics A gene. The suicideplasmid vector, used, contains the ics A gene of S. flexneri (60, 61),that has a significant deletion at a restriction site, into which isinserted a fragment coding for resistance to cadmium (63, 64) and asuitable promoter for the cadmium-resistance gene. The resulting Tox A⁻,Ent⁻, ics A⁻ S. dysenteriae 1 clone is characterized by a substantiallyreduced invasiveness, which renders it suitable for making a vaccine forhumans against S. dysenteriae 1.

It is believed that this invention and many of its attendant advantageswill be understood from its description above, and it will be apparentthat various modifications can be made in the method and vaccinedescribed above without departing from the spirit and scope of theinvention or sacrificing all of its material advantages, the embodimentsdescribed above being merely preferred embodiments.

The references, referred to above, are as follows.

REFERENCES

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TABLE 1 Strains, plasmids, phages and their relevant characteristicsPlasmid/ Strain Species Genotype phage Relevant characteristics SC 500S. dysentariae 1 thi, nad, trp, met pHS7200 Invasion of HeLa cells SC501 S. dysenteriae 1 thi, nad, trp, met, tox, spc^(r) pHS7200 Invasionof HeLa cells SC 502 S. dysentariae 1 thi, nad, trp, met — — SC 503 S.dysenteriae 1 thi, nad, trp, met, tox, spc^(r) — — Y 1089 E. coliΔlacU169 proA⁺ Δlon araD139 pHC9 Ap^(r), pBR322-lac i^(q) strA hflΔl50[chr::Tn10] λGT11 lac5Δ(shindIIIλ2–3) srIλ3^(φ) cI857 arIλ4′ nin5arIλ5^(α) sam100 Y 1090 E. coli ΔlacU169 proA⁺ Δlon araD139 pHC9 Ap^(r),pBR322-lac i^(q) strA supF[trpC22::Tn10] JM 83 E. coli F⁻, araΔlac-prostrA pUC8 Ap^(r), cloning vehicle thi, phi80dlacZ AM15 pHS7201 Ap^(r),Shiga toxin genes subcloned in pUC8 pHS7202 Ap^(r) Spc^(r) Ω is insertedat the HpaI site of pHS6001 pHP45 Ap^(r) Spe^(r) contains the Ω elementSM10λpir E. coli recA, RP4-2 TC::Mu Km^(r) λpir contains the pir thi,thr, leu, suIII function from R6K replication origin pJM703-1 Suicidecloning vector Ap^(r), can be mobilized in SM10λpir pHS7203 Mutagenizedtoxin genes cloned in pJM703-1 Ap^(r) Sp^(r) HB101 E. coli RB⁻, MB⁻,recA, supE44 — — (su2)lacY, leuB6, proA2 thi-1 Sm^(r)

1. A method for modifying a wild strain of an enteroinvasive Shigella toproduce a modified strain of Shigella that can not spread substantiallywithin infected cells of a host and can not spread substantially frominfected to uninfected cells of the host, for use in making a vaccineagainst the wild strain of Shigella, the method comprising inactivatingan icsA gene of the wild strain of Shigella, other than only byinactivation by means of a transposon inserted into the gene, to therebyprovide a modified strain of Shigella that can not spread substantiallywithin infected cells of the host and can not spread substantially frominfected to uninfected cells of the host.
 2. The method of claim 1,wherein the modified strain of Shigella also can not substantiallyinvade cells of the host, the method further comprising inactivating anaerobactin or enterochelin gene of the wild strain of Shigella, otherthan only by inactivation by means of a transposon inserted into thegene, to thereby provide a modified strain of Shigella that can notspread substantially within infected cells of the host, can not spreadsubstantially from infected to uninfected cells of the host, and can notsubstantially invade cells of the host.
 3. The method of claim 2,wherein the modified strain of Shigella also can not produce toxins thatkill a substantial number of the host's cells, the method furthercomprising inactivating a Shiga-toxin gene of the wild strain ofShigella, other than only by inactivation by means of a transposoninserted into the gene, to thereby provide a modified strain of Shigellathat can not spread substantially within infected cells of the host, cannot spread substantially from infected to uninfected cells of the host,can not substantially invade cells of the host, and can not producetoxins that kill a substantial number of the host's cells.
 4. The methodof any of claims 1–3, wherein said Shigella is S. flexneri.
 5. Themethod of any of claims 1–3, wherein said Shigella is S. dysenteriae 1.6. The method of claim 5, wherein one or more of the ent F, Fep E, FepC, and Fep D subunit genes of the enterochelin operon of S. dysenteriae1 are modified.
 7. The method of claim 3, wherein the Shiga-toxin geneis the Shiga-toxin A gene.
 8. The method of any of claims 1–3, whereinone or more of said inactivated genes are inactivated genes from whichat least one nucleotide sequence has been deleted.
 9. The method of anyof claims 1–3, wherein one or more of said inactivated genes areinactivated genes into which at least one nucleotide sequence has beeninserted.
 10. The method of claim 9, wherein a marker gene is insertedinto one or more of said inactivated genes.
 11. The method of claim 3,further comprising isolating said modified strain of Shigella from saidwild strain of Shigella.
 12. A modified Shigella for use in making avaccine against a wild strain of Shigella, the modified Shigellacomprising: (a) an inactivated icsA gene, inactivated other than only bymeans of a transposon inserted into the gene; and (b) an inactivatedaerobactin or enterochelin gene, inactivated other than only by means ofa transposon inserted into the gene; wherein the modified Shigella cannot spread substantially within infected cells of the host, can notspread substantially from infected to uninfected cells of the host, andcan not substantially invade cells of the host.
 13. The Shigella ofclaim 12, further comprising an inactivated Shiga-toxin gene,inactivated other than only by means of a transposon inserted into thegene; wherein the modified Shigella can not spread substantially withininfected cells of the host, can not spread substantially from infectedto uninfected cells of the host, can not substantially invade cells ofthe host, and can not produce toxins that kill a substantial number ofthe host's cells.
 14. The Shigella of claim 13, wherein the Shiga-toxingene is Shiga-toxin A.
 15. The Shigella of claim 12 or 13, wherein saidShigella is S. dysenteriae 1 or S. flexneri.
 16. The Shigella of claim12 or 13, comprising inactivated ent F, Fep E, Fep C, or Fep D subunitgenes of the enterochelin operon.
 17. The Shigella of claim 12 or 13,wherein one or more of said inactivated genes are inactivated genes fromwhich at least one nucleotide sequence has been deleted.
 18. TheShigella of claim 12 or 13 wherein one or more of said inactivated genesare inactivated genes into which at least one nucleotide sequence hasbeen inserted.
 19. The Shigella of claim 18 wherein a marker gene isinserted into one or more of said inactivated genes.
 20. A vaccinecomprising the Shigella of claim 12 or 13 and a pharmaceuticallyacceptable vehicle.
 21. The method of any of claims 1–3, wherein amarker gene is inserted into each inactivated gene.
 22. The Shigella ofclaim 12 or 13, wherein a marker gene is inserted into each inactivatedgene.
 23. A vaccine comprising the Shigella of claim 22 and apharmaceutically acceptable vehicle.
 24. The method of claim 1, whereinsaid inactivation of said icsA gene comprises allelic exchange with amutagenized icsA gene that has been mutagenized in vitro.
 25. The methodof claim 2, wherein said inactivation of said icsA gene comprisesallelic exchange with a mutagenized icsA gene that has been mutagenizedin vitro, and wherein said inactivation of said aerobactin orenterochelin gene comprises allelic exchange with a mutagenizedaerobactin or enterochelin gene that has been mutagenized in vitro. 26.The method of claim 3, wherein said inactivation of said icsA genecomprises allelic exchange with a mutagenized icsA gene that has beenmutagenized in vitro, wherein said inactivation of said aerobactin orenterochelin gene comprises allelic exchange with a mutagenizedaerobactin or enterochelin gene that has been mutagenized in vitro, andwherein said inactivation of said Shiga-toxin gene comprises allelicexchange with a mutagenized Shiga-toxin gene that has been mutagenizedin vitro.
 27. The method of any of claims 24–26, wherein a marker geneis inserted into one or more of said mutagenized genes.
 28. A modifiedShigella for use in making a vaccine against a wild strain of Shigella,the modified Shigella comprising: (a) an inactivated icsA gene,inactivated by allelic exchange with a mutagenized icsA gene that hasbeen mutagenized in vitro, wherein said mutagenesis is other than onlyby means of a transposon inserted into the gene; and (b) an inactivatedaerobactin or enterochelin gene, inactivated by allelic exchange with amutagenized aerobactin or enterochelin gene that has been mutagenized invitro, wherein said mutagenesis is other than only by means of atransposon inserted into the gene; wherein the modified Shigella can notspread substantially within infected cells of the host, can not spreadsubstantially from infected to uninfected cells of the host, and can notsubstantially invade cells of the host.
 29. The Shigella of claim 28,further comprising an inactivated Shiga-toxin gene, inactivated byallelic exchange with a mutagenized Shiga-toxin gene that has beenmutagenized in vitro, wherein said mutagenesis is other than only bymeans of a transposon inserted into the gene; wherein the modifiedShigella can not spread substantially within infected cells of the host,can not spread substantially from infected to uninfected cells of thehost, can not substantially invade cells of the host, and can notproduce toxins that kill a substantial number of the host's cells. 30.The Shigella of claim 28 or 29, wherein a marker gene is inserted intoone or more of said mutagenized genes.
 31. A vaccine comprising theShigella of claim 28 or 29 and a pharmaceutically acceptable vehicle.32. A modified Shigella for use in making a vaccine against a wildstrain of Shigella, the modified Shigella comprising an inactivated icsAgene, inactivated other than only by means of a transposon inserted intothe gene; wherein the modified Shigella can not spread substantiallywithin infected cells of the host and can not spread substantially frominfected to uninfected cells of the host.
 33. The Shigella of claim 32,wherein at least one nucleotide sequence has been deleted from saidinactivated icsA gene.
 34. The Shigella of claim 32, wherein at leastone nucleotide sequence has been inserted into said inactivated icsAgene.
 35. The Shigella of claim 34, wherein a marker gene is insertedinto said inactivated icsA gene.
 36. A vaccine comprising the Shigellaof claim 32 and a pharmaceutically acceptable vehicle.
 37. A modifiedShigella for use in making a vaccine against a wild strain of Shigella,the modified Shigella comprising an inactivated icsA gene, inactivatedby allelic exchange with a mutagenized icsA gene that has beenmutagenized in vitro, wherein said mutagenesis is other than only bymeans of a transposon inserted into the gene; wherein the modifiedShigella can not spread substantially within infected cells of the hostand can not spread substantially from infected to uninfected cells ofthe host.
 38. The Shigella of claim 37, wherein a marker gene isinserted into said mutagenized icsA gene.
 39. A vaccine comprising theShigella of claim 37 and a pharmaceutically acceptable vehicle.